IL273119B1 - Constrained conditionally activated binding proteins - Google Patents
Constrained conditionally activated binding proteinsInfo
- Publication number
- IL273119B1 IL273119B1 IL273119A IL27311920A IL273119B1 IL 273119 B1 IL273119 B1 IL 273119B1 IL 273119 A IL273119 A IL 273119A IL 27311920 A IL27311920 A IL 27311920A IL 273119 B1 IL273119 B1 IL 273119B1
- Authority
- IL
- Israel
- Prior art keywords
- domain
- seq
- pseudo
- sdabd
- polypeptide
- Prior art date
Links
- 102000014914 Carrier Proteins Human genes 0.000 title description 5
- 108091008324 binding proteins Proteins 0.000 title description 5
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 76
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 76
- 229920001184 polypeptide Polymers 0.000 claims description 75
- 230000027455 binding Effects 0.000 claims description 52
- 108090000623 proteins and genes Proteins 0.000 claims description 52
- 102000004169 proteins and genes Human genes 0.000 claims description 50
- 206010028980 Neoplasm Diseases 0.000 claims description 43
- 239000000427 antigen Substances 0.000 claims description 41
- 102000036639 antigens Human genes 0.000 claims description 41
- 108091007433 antigens Proteins 0.000 claims description 41
- 210000004027 cell Anatomy 0.000 claims description 32
- 102000035195 Peptidases Human genes 0.000 claims description 28
- 108091005804 Peptidases Proteins 0.000 claims description 28
- 239000004365 Protease Substances 0.000 claims description 28
- 210000004899 c-terminal region Anatomy 0.000 claims description 28
- 239000000203 mixture Substances 0.000 claims description 27
- 108020004707 nucleic acids Proteins 0.000 claims description 20
- 102000039446 nucleic acids Human genes 0.000 claims description 20
- 150000007523 nucleic acids Chemical class 0.000 claims description 20
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims description 19
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims description 19
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 claims description 18
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 claims description 17
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 claims description 17
- 239000013604 expression vector Substances 0.000 claims description 14
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 claims description 12
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 claims description 12
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 10
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 8
- 101001023230 Homo sapiens Folate receptor alpha Proteins 0.000 claims description 7
- 201000011510 cancer Diseases 0.000 claims description 7
- 102100035139 Folate receptor alpha Human genes 0.000 claims description 6
- 102100038078 CD276 antigen Human genes 0.000 claims description 5
- 101000884279 Homo sapiens CD276 antigen Proteins 0.000 claims description 5
- 108010091175 Matriptase Proteins 0.000 claims description 4
- 102100037942 Suppressor of tumorigenicity 14 protein Human genes 0.000 claims description 4
- 108090000190 Thrombin Proteins 0.000 claims description 4
- 102100026802 72 kDa type IV collagenase Human genes 0.000 claims description 3
- 108090000625 Cathepsin K Proteins 0.000 claims description 3
- 102000004171 Cathepsin K Human genes 0.000 claims description 3
- 108090000613 Cathepsin S Proteins 0.000 claims description 3
- 102100035654 Cathepsin S Human genes 0.000 claims description 3
- 101000627872 Homo sapiens 72 kDa type IV collagenase Proteins 0.000 claims description 3
- 108090000265 Meprin A Proteins 0.000 claims description 3
- 102100030876 Meprin A subunit beta Human genes 0.000 claims description 3
- 108090000263 Meprin B Proteins 0.000 claims description 3
- 229960004072 thrombin Drugs 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims 4
- 238000004519 manufacturing process Methods 0.000 claims 4
- 108090000624 Cathepsin L Proteins 0.000 claims 2
- 102000004172 Cathepsin L Human genes 0.000 claims 2
- -1 GranzymeB Proteins 0.000 claims 2
- 102100034867 Kallikrein-7 Human genes 0.000 claims 2
- 101710176222 Kallikrein-7 Proteins 0.000 claims 2
- 102100031358 Urokinase-type plasminogen activator Human genes 0.000 claims 2
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 claims 2
- 235000018102 proteins Nutrition 0.000 description 46
- 238000003776 cleavage reaction Methods 0.000 description 41
- 230000007017 scission Effects 0.000 description 41
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 31
- 150000001413 amino acids Chemical class 0.000 description 27
- 235000001014 amino acid Nutrition 0.000 description 23
- 230000000694 effects Effects 0.000 description 21
- 229940002612 prodrug Drugs 0.000 description 19
- 239000000651 prodrug Substances 0.000 description 19
- 230000006035 T cell-directed cellular cytotoxicity Effects 0.000 description 14
- 230000008685 targeting Effects 0.000 description 11
- 238000003556 assay Methods 0.000 description 10
- 239000013641 positive control Substances 0.000 description 10
- 239000013642 negative control Substances 0.000 description 7
- 238000006467 substitution reaction Methods 0.000 description 7
- 210000001744 T-lymphocyte Anatomy 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 230000009870 specific binding Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 241000270295 Serpentes Species 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 102000007562 Serum Albumin Human genes 0.000 description 3
- 108010071390 Serum Albumin Proteins 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 108091007169 meprins Proteins 0.000 description 3
- 238000009877 rendering Methods 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 102000001708 Protein Isoforms Human genes 0.000 description 2
- 108010029485 Protein Isoforms Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 230000009830 antibody antigen interaction Effects 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 235000009697 arginine Nutrition 0.000 description 2
- 150000001720 carbohydrates Chemical group 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000003292 diminished effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000001338 self-assembly Methods 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000661807 Homo sapiens Suppressor of tumorigenicity 14 protein Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 1
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 1
- NPPQSCRMBWNHMW-UHFFFAOYSA-N Meprobamate Chemical compound NC(=O)OCC(C)(CCC)COC(N)=O NPPQSCRMBWNHMW-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 206010054094 Tumour necrosis Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 102000025171 antigen binding proteins Human genes 0.000 description 1
- 108091000831 antigen binding proteins Proteins 0.000 description 1
- 238000012754 cardiac puncture Methods 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- PQYUGUXEJHLOIL-UHFFFAOYSA-N diethoxysilyl triethyl silicate Chemical compound C(C)O[SiH](O[Si](OCC)(OCC)OCC)OCC PQYUGUXEJHLOIL-UHFFFAOYSA-N 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 102000006815 folate receptor Human genes 0.000 description 1
- 108020005243 folate receptor Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 150000002333 glycines Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 108010021083 hen egg lysozyme Proteins 0.000 description 1
- 102000053180 human FOLR1 Human genes 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000012642 immune effector Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000004882 non-tumor cell Anatomy 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000004400 serine Nutrition 0.000 description 1
- 150000003355 serines Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
- C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oncology (AREA)
- Biomedical Technology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
118459-5005-WO CONSTRAINED CONDITIONALLY ACTIVATED BINDING PROTEINS I. CROSS REFERENCE TO RELATED APPLICATIONS id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1"
id="p-1"
[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application 62/555,943 filed September 8, 2017, U.S. Provisional Patent Application 62/586,627 filed November 15, 2017, and U.S. Provisional Patent Application 62/587,318 filed November 16, 2017, all of which are expressly incorporated herein by reference in their entirety.
II. REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK. id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2"
id="p-2"
[0002] The sequence listing contained in the file named "118459-5005_ST25.txt"and having a size of 984 kilobytes, has been submitted electronically herewith via EFS-Web, and the contents of the txt file are hereby incorporated by reference in their entirety.
III. BACKGROUND OF THE INVENTION id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3"
id="p-3"
[0003] The selective destruction of an individual cell or a specific cell type is often desirable in a variety of clinical settings. For example, it is a primary goal of cancer therapy to specifically destroy tumor cells, while leaving healthy cells and tissues as intact and undamaged as possible. One such method is by inducing an immune response against the tumor, to make immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs) attack and destroy tumor cells. id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4"
id="p-4"
[0004] The use of intact monoclonal antibodies (mAb), which provide superior binding specificity and affinity for a tumor-associated antigen, have been successfully applied in the area of cancer treatment and diagnosis. However, the large size of intact mAbs, their poor bio-distribution, low potency and long persistence in the blood pool have limited their clinical applications. For example, intact antibodies can exhibit specific accumulation within the tumor area. In biodistribution studies, an inhomogeneous antibody distribution with 118459-5005-WO primary accumulation in the peripheral regions is noted when precisely investigating the tumor. Due to tumor necrosis, inhomogeneous antigen distribution and increased interstitial tissue pressure, it is not possible to reach central portions of the tumor with intact antibody constructs. In contrast, smaller antibody fragments show rapid tumor localization, penetrate deeper into the tumor, and also, are removed relatively rapidly from the bloodstream. However, many antibodies, including scFvs and other constructs, show "on target/off tumor" effects, wherein the molecule is active on non-tumor cells, causing side effects, some of which can be toxic. The present invention is related to novel constructs that are selectively activated in the presence of tumor proteases.
IV. SUMMARY OF THE INVENTION id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5"
id="p-5"
[0005] The present invention provides a number of different protein compositions for the treatment of cancer. Accordingly, in one aspect, the invention provides "Format 2" proteins comprising, from N- to C-terminal: a first single domain antigen binding domain (sdABD) that binds to a human tumor target antigen (TTA) (sdABD-TTA); b) a first domain linker; c) a constrained Fv domain comprising: i) a first variable heavy domain comprising a vhCDR1, vhCDR2 and vhCDR3; ii) a constrained non-cleavable linker (CNCL); and iii) a first variable light domain comprising vlCDR1, vlCDR2 and vlCDR3; d) a second domain linker; e) a second sdABD-TTA; f) a cleavable linker (CL); g) a constrained pseudo Fv domain comprising: i) a first pseudo light variable domain; ii) a non-cleavable linker (NCL); and iii) a first pseudo heavy variable domain; h) a third domain linker; and i) a third sdABD that binds to human serum albumin; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 but said constrained Fv domain does not bind CD3; said first variable heavy domain and said first pseudo variable light domain intramolecularly associate to form an inactive Fv; and said first variable light domain and said first pseudo variable heavy domain intramolecularly associate to form an inactive Fv. id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6"
id="p-6"
[0006] In a further aspect, the invention provides "Format 1" proteins comprising, from N- to C-terminal: a) a first sdABD-TTA; b) a first domain linker; c) a constrained Fv domain 118459-5005-WO comprising: i) a first variable heavy domain comprising a vhCDR1, vhCDR2 and vhCDR3; ii) a constrained cleavable linker (CCL); and iii) a first variable light domain comprising vlCDR1, vlCDR2 and vlCDR3; d) a second domain linker; e) a second sdABD-TTA; f) a cleavable linker (CL); g) a constrained pseudo Fv domain comprising: i) a first pseudo light variable domain; ii) a non-cleavable linker (NCL); and iii) a first pseudo heavy variable domain; h) a third domain linker; and i) a third sdABD that binds to human serum albumin; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 but said constrained Fv domain does not bind CD3; wherein said first variable heavy domain and said first pseudo variable light domain intramolecularly associate to form an inactive Fv; and wherein said first variable light domain and said first pseudo variable heavy domain intramolecularly associate to form an inactive Fv.
In an additional aspect, the invention provides "Format 4" proteins comprising, from N- to C-terminal: a) a single domain antigen binding domain (sdABD) that binds to a human tumor target antigen (TTA) (sdABD-TTA); b) a first domain linker; c) a constrained Fv domain comprising: i) a first variable heavy domain comprising a vhCDR1, vhCDR2 and vhCDR3; ii) a constrained non-cleavable linker (CNCL); and iii) a first variable light domain comprising vlCDR1, vlCDR2 and vlCDR3; d) a cleavable linker (CL); e) a second sdABD that binds to human serum albumin; f) a domain linker; g) a constrained pseudo Fv domain comprising: i) a first pseudo light variable domain; ii) a non-cleavable linker (NCL); and iii) a first pseudo heavy variable domain; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 but said constrained Fv domain does not bind CD3; wherein said first variable heavy domain and said first pseudo variable light domain intramolecularly associate to form an inactive Fv; and wherein said first variable light domain and said first pseudo variable heavy domain intramolecularly associate to form an inactive Fv. id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7"
id="p-7"
[0007] In a further aspect to the Format 1, Format 2 and Format 4 proteins listed above, said first variable heavy domain is N-terminal to said first variable light domain and said pseudo light variable domain is N-terminal to said pseudo variable heavy domain. 118459-5005-WO id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8"
id="p-8"
[0008] In a further aspect to the Format 1, Format 2 and Format 4 proteins listed above, said first variable heavy domain is N-terminal to said first variable light domain and said pseudo variable heavy domain is N-terminal to said pseudo variable light domain. id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9"
id="p-9"
[0009] In a further aspect to the Format 1, Format 2 and Format 4 proteins listed above, said first variable light domain is N-terminal to said first variable heavy domain and said pseudo light variable domain is N-terminal to said pseudo variable heavy domain. id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10"
id="p-10"
[0010] In a further aspect to the Format 1, Format 2 and Format 4 proteins listed above, said first variable light domain is N-terminal to said first variable heavy domain and said pseudo variable heavy domain is N-terminal to said pseudo variable light domain. id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11"
id="p-11"
[0011] In an additional aspect, the invention provides Format 1 and 2 proteins wherein said first and second TTA are the same. id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12"
id="p-12"
[0012] In a further aspect, the invention provides Format 1 and 2 proteins wherein said first and second TTA are different. id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13"
id="p-13"
[0013] In an additional aspect, the invention provides Format 1, 2 and 4 proteins wherein said first and second TTA are selected from EGFR, EpCAM, FOLR1 and B7H3. These sequences can be selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29; SEQ ID NO:33; SEQ ID NO:37 and SEQ ID NO:41. id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14"
id="p-14"
[0014] In a further aspect, the invention provides Format 1, 2 and 4 proteins wherein said half-life extension domain has SEQ ID NO:45. id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15"
id="p-15"
[0015] In an additional aspect, the invention provides Format 1, 2 and 4 proteins wherein said cleavable linker is cleaved by a human protease selected from the group consisting of MMP2, MMP9, Meprin A, Meprin B, Cathepsin S, Cathepsin K, Cathespin L, GranzymeB, uPA, Kallekriein7, matriptase and thrombin. id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16"
id="p-16"
[0016] In a further aspect, the invention provides a protein selected from the group consisting of Pro186, Pro225, Pro226, Pro233, Pro311, Pro312, Pro313, Pro495, Pro246, Pro254, Pro255, Pro256, Pro420, Pro421, Pro432, Pro479, Pro480, Pro187, Pro221, Pro222, Pro223, Pro224, Pro393, Pro394, Pro395, Pro396, Pro429, Pro430 and Pro431. 118459-5005-WO id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17"
id="p-17"
[0017] In an additional aspect, the invention provides nucleic acids encoding a Format 1, Format 2 or Format 4 protein as described herein, as well as expression vectors and host cells comprising the nucleic acids encoding the protein. id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18"
id="p-18"
[0018] In a further aspect, the invention provides methods of making the proteins of the invention and methods of treating patients in need thereof. id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19"
id="p-19"
[0019] In an additional aspect, the invention provides compositions comprising "Format 3A" pairs of pro-drug proteins, comprising: a) a first protein comprising, from N- to C-terminal: i) a first sdABD-TTA; ii) a first domain linker; iii) a pseudo Fv domain comprising, from N- to C-terminal: 1) a variable heavy chain comprising a vhCDR1, vhCDR2 and vhCDR3; 2) a cleavable linker; and 3) a first pseudo variable light domain comprising iVLCDR1, iVLCDR2 and iVLCDR3; iv) a second domain linker; v) a sdABD-HSA; a) a first second protein comprising, from N- to C-terminal: i) a third sdABD that binds to a human tumor target antigen; ii) a third domain linker; iii) a pseudo Fv domain comprising, from N- to C-terminal: 1) a variable light chain comprising a VLCDR1, VLCDR2 and VLCDR3; 2) a cleavable linker; and 3) a first pseudo variable heavy domain comprising iVHCDR1, iVHCDR2 and iVHCDR3; iv) a fourth domain linker; v) a sdABD-HSA;; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 when associated; wherein said first variable heavy domain and said first pseudo variable light domain intermolecularly associate to form an inactive Fv; wherein said first variable light domain and said first pseudo variable heavy domain intermolecularly associate to form an inactive Fv; and wherein said first and third sdABD are selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29; SEQ ID NO:33; SEQ ID NO:37 and SEQ ID NO:41. id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20"
id="p-20"
[0020] In a further aspect, the invention provides compositions comprising "Format 3B" pairs of pro-drug proteins, comprising a) a first protein comprising, from N- to C-terminal: i) a first sdABD-TTA; ii) a first domain linker; iii) a second sdABD-TTA; iv) a second domain linker; iii) a pseudo Fv domain comprising, from N- to C-terminal: 1) a variable heavy chain comprising a vhCDR1, vhCDR2 and vhCDR3; 2) a cleavable linker; and 3) a first pseudo variable light domain comprising iVLCDR1, iVLCDR2 and iVLCDR3; iv) a third domain 118459-5005-WO linker; and v) a sdABD-HSA; a) a first second protein comprising, from N- to C-terminal: i) a third sdABD-TTA; ii) a fourth domain linker; iii) a fourth sdABD-TTA; iv) a fifth domain linker; iii) a pseudo Fv domain comprising, from N- to C-terminal: 1) a variable light chain comprising a VLCDR1, VLCDR2 and VLCDR3; 2) a cleavable linker; and 3) a first pseudo variable heavy domain comprising iVHCDR1, iVHCDR2 and iVHCDR3; iv) a sixth domain linker; v) a sdABD-HSA; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 when associated; wherein said first variable heavy domain and said first pseudo variable light domain intermolecularly associate to form an inactive Fv; and wherein said first variable light domain and said first pseudo variable heavy domain intermolecularly associate to form an inactive Fv. id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21"
id="p-21"
[0021] In an additional aspect, Format 3A and Format 3B proteins have sdABD-HSA that have SEQ ID NO:45. id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22"
id="p-22"
[0022] In a further aspect, Format 3A and Format 3B proteins have sdABD-TTA that binds to a TTA selected from EGFR, EpCAM, FOLR1 and B7H3. The sdABD-TTAs can be selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29; SEQ ID NO:33; SEQ ID NO:37 and SEQ ID NO:41. id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23"
id="p-23"
[0023] In a further aspect, the invention provides nucleic acid compositions comprising first nucleic acids that encode the first protein members of the prodrug pair and second nucleic acids that encode the second protein members of the pairs, and expression vectors and host cells containing the nucleic acids. V. BRIEF DESCRIPTION OF THE DRAWINGS id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24"
id="p-24"
[0024] Figure 1 depicts the "format 1" type of protease activation of the present invention, referred to herein as "constrained, cleavable constructs" or "cc constructs". In this embodiment, a representative construct is Pro140: there are ABDs for two TTA (as depicted in Figure 1, these are both the same, although as described herein they can be different). Upon cleavage, the prodrug construct splits into three components, one containing an α-TTA domain linked via a domain linker to an active VH of αCD3, the second containing an α-TTA domain linked via a domain linker to an active VL of αCD3, and a "leftover" piece 118459-5005-WO comprising the half life extension domain linked to the inactive VH and VL. The two active variable domains then are free to associate to form a functional anti-CD3 binding domain. It should be noted that in "format 1" embodiments, the resulting active component is trivalent: there is monovalent binding to CD3 and bivalent binding to the TTA, rendering a bispecific binding protein, although in some cases this trivalency could be trispecifics, with monovalent binding to CD3, monovalent binding to a first TTA and monovalent binding to a second TTA. Figure 1 also shows an anti-human serum albumin (HSA) domain as a half-life extension domain, in many embodiments an sdABD as defined herein, although as discussed herein, this is optional and/or can be replaced by other half-life extension domains; additionally, the half-life extension domain can also be N-terminal to the construct or internal as well. Figure 1 also has the VH and VL of the Fv and iVH and iVL of the pseudo Fv in a specific order, e.g. from N- to C-terminal, VH-linker-VL (and iVL-linker-iVH) although as will be appreciated by those in the art, these can be reversed (VL-linker-VH and iVH-linker-iVL). Alternatively, one of these Fvs can be in one orientation and the other in the other orientation, although the expression of protein in the orientation as shown here was surprisingly higher than the other orientations. id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25"
id="p-25"
[0025] Figure 2 depicts the "format 2" type of protease activation of the present invention, referred to herein as "constrained, non-cleavable constructs", or "CNCL constructs", also sometimes referred to herein as "dimerization constructs" as discussed herein. These constructs do not isomerize as discussed herein. Upon cleavage, two prodrug construct splits into four components, two half-life extension domains (in this case, sdABDs to HSA) linked to two pseudo domains (which may or may not be able to self-associate, depending on the length of the linkers and the inactivating mutations), and two active moieties that self-assemble into a dimeric active moiety that contains four anti-TTA domains (which can be all the same or two are the same and the other two are different). It should be noted that in "format 2" embodiments, the resulting active component is hexavalent: there is bivalent binding to CD3 and quadrivalent binding to the TTA, rendering a bispecific binding protein, although in some cases this hexavalency could be trispecifics, with bivalent binding to CD3, bivalent binding to a first TTA and bivalent binding to a second TTA. Figure 2 also shows an anti-human serum albumin (HSA) domain as a half-life extension domain, in many embodiments an sdABD as defined herein, although as discussed herein, this is optional 118459-5005-WO and/or can be replaced by other half-life extension domains; additionally, the half-life extension domain can also be N-terminal to the construct or internal as well. Figure 2 also has the VH and VL of the Fv and iVH and iVL of the pseudo Fv in a specific order, e.g. from N- to C-terminal, VH-linker-VL (and iVL-linker-iVH) although as will be appreciated by those in the art, these can be reversed (VL-linker-VH and iVH-linker-iVL). Alternatively, one of these Fvs can be in one orientation and the other in the other orientation, although the expression of protein in the orientation as shown here was surprisingly higher than the other orientations. id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26"
id="p-26"
[0026] Figure 3A - Figure 3B depict "format 3" type of constructs, also sometimes referred to as "hemi-constructs" or "hemi-COBRA™" as outlined herein, as these are two different polypeptide chains that together make up an MCE therapeutic as is further discussed herein. In this embodiment, the constructs are delivered in pairs, with the pre-cleavage intramolecular self-assembly resulting in inactive anti-CD3 Fv domains. Upon cleavage, the inert variable domains are released, and the two active variable domains then intermolecularly assemble, to form an active anti-CD3 binding domain. The two sdABD-TTAs bind to the corresponding receptor on the tumor cell surface, and the cleavage is done by a protease. This allows the intermolecular assembly, since the molecules are physically held in place, favoring the assembly of the active anti-CD3 domain. As above for formats and 2, in this embodiment, the N- to C-terminal order of the variable domains can be reversed, or mixed as well. Furthermore, the sdABD(HSA) can be either at the N- or C-terminus of each hemi-construct. Pro16 has the sdABD(HSA) at the C terminus and Prohas it at the N-terminus (see Pro19, SEQ ID NO:XX, has the sdABD(HSA) at the C-terminus). Figure 3A shows Format 3 constructs with a single sdABD-TTA domain per hemi-construct, and Figure 3B shows Format 3 constructs with two sdABD-TTAs per hemi-construct, in a "dual targeting" or "hetero-targeting" format. Note that Figure 3B uses FOLR1 and EGFR as the two TTAs, but other combinations as outlined herein can also be used. id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27"
id="p-27"
[0027] Figure 4 depicts "format 4" type of constructs that are similar to "format 2" constructs but have only a single sdABD-TTA. The figure shows the sdABD-TTA to EGFR, but as will be appreciated by those in the art, other TTA can be used as well. Upon cleavage, the prodrug construct splits into two components, a half-life extension domain (in this case, sdABDs to HSA) linked to a pseudo Fv and an active moiety, that in the presence of a second 118459-5005-WO active moiety from a different cleaved molecule, self-assembles into a dimeric active moiety that contains two anti-TTA domains. It should be noted that in "format 4" embodiments, the resulting active component is quadrivalent: there is bivalent binding to CD3 and bivalent binding to the TTA, rendering a bispecific binding protein. Figure 4 also shows an anti-human serum albumin (HSA) domain as a half-life extension domain, in many embodiments an sdABD(½) as defined herein, although as discussed herein, this is optional and/or can be replaced by other half-life extension domains; additionally, the half-life extension domain can also be N-terminal to the construct or internal as well. Figure 4 also has the VH and VL of the Fv and iVH and iVL of the pseudo Fv in a specific order, e.g. from N- to C-terminal, VH-linker-VL (and iVL-linker-iVH) although as will be appreciated by those in the art, these can be reversed (VL-linker-VH and iVH-linker-iVL). Alternatively, one of these Fvs can be in one orientation and the other in the other orientation, although the expression of protein in the orientation as shown here was surprisingly higher than the other orientations. id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28"
id="p-28"
[0028] Figure 5A – Figure 5G depict a number of sequences of the invention. For antigen binding domains, the CDRs are underlined. As is more fully outlined herein, these domains can be assembled in a wide variety of configurations in the present invention, including "format 1", "format 2", "format 3" and "format 4" orientations. Of note is that SEQ ID NO:90 is cleaved by MMP9 slightly faster than SEQ ID NO:s 75 and 76, and SEQ ID NO:is cleaved slower than SEQ ID NO:s 75 and 76. id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29"
id="p-29"
[0029] Figure 6A – Figure 6B depict a number of suitable protease cleavage sites. As will be appreciated by those in the art, these cleavage sites can be used as cleavable linkers. In some embodiments, for example when more flexible cleavable linkers are required, there can be additional amino acids (generally glycines and serines) that are either or both N- and C-terminal to these cleavage sites. id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30"
id="p-30"
[0030] Figure 7A – Figure 7D depict some data associated with the "Format 3" or "hemi-COBRA™" structures. This shows that Format 3 constructs bind co-operatively to CD3 after cleavage by protease (in this case EK protease, although any of the protease cleavage sites outlined herein and depicted in Figure 5 and Figure 6 can be used) and create a CD3 binding site, as shown by a sandwich FACS analysis. id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31"
id="p-31"
[0031] Figure 8A – Figure 8D shows that the protease cleavage co-operatively activates T-cell killing of EGFR+ target cells with complimentary hemi-COBRA™ pairs. Figure 8A and Figure 8B show that the constructs in isolation, but cleaved with different concentrations of 118459-5005-WO protease, do not affect target cell viability. However, Figure 8C shows that in combination, in the presence of protease, target cell viability is significantly diminished. Figure 8D shows the general mechanism. id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32"
id="p-32"
[0032] Figure 9 shows some non-target controls for use in the assays to test efficacy of the Format 1 constructs. id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33"
id="p-33"
[0033] Figure 10A – Figure 10F shows the generation of an active CD3 binding domain is dependent on target binding of both "arms", e.g. the sdABD-TTA domains, one of which is on each of the two constructs. The TDCC assay was done as described in the Examples. id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34"
id="p-34"
[0034] Figure 11 shows the schematic of suitable hemi-COBRA™ pairs. "Mep" stands for a meprin protease cleavage site, "His-6" is a tag as more fully discussed herein, ST14 is a matriptase protease cleavage site and "Thb" is a thrombin protease cleavage site. id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35"
id="p-35"
[0035] Figure 12A – Figure 12C shows the TDCC data associated with the constructs of Figure 11. Figure 12A shows that addition of pre-cleaved hemi-COBRA pairs results in efficacy on OvCAR8 cells, Figure 12B shows that addition of pre-cleaved hemi-COBRA pairs results in efficacy on HCT116 cells, and Figure 12C shows that addition of pre-cleaved hemi-COBRA pairs results in efficacy on LoVo cells, all of which are cancer cell lines. id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36"
id="p-36"
[0036] Figure 13A – Figure 13B shows that the MMP9 linker is stable in vivo. NSG mice were administered a single intravenous bolus dose of either Pro40 (MMP9 cleavable), Pro(non-cleavable) via the tail vein at a dose level of 0.5 mg/kg. The dose solution for each compound was prepared in a vehicle of 25mM citric acid, 75-mM L-arginine, 75mM NaCl and 4% sucrose pH 7.0. Two blood samples were collected at preselected times from each animal, one towards the beginning of the study, collected by orbital bleed or submandibular bleed, and another at the terminal time point by cardiac puncture. The time points for blood collection were 0.083, 1, 6, 24, 72, and 168h. Plasma was prepared from each individual blood sample using K 2 EDTA tubes. Concentrations were determined using an MSD assay with a MAb specific to the anti-HSA sdABD and detected with the EGFR extracellular domain. id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37"
id="p-37"
[0037] Figure 14 depicts the schematics of the Format 3A hemi-COBRA™ constructs used in the experiments depicted in Figure]] 15. Pro51 is the positive control, as it is "always on", since it forms an active anti-CD3 Fv. Pro98 is a negative control, since it’s sdABD is directed against hen egg lysozyme, which isn’t expressed by the tumor. Pro77 and Pro53 are 118459-5005-WO the pro-drug Format 3A pair, using sdABDs against EGFR and an MMP9 cleavage site. Pro74 and Pro72 is a negative control Format 3A pair, since they don’t have cleavage sites. id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38"
id="p-38"
[0038] Figure 15 shows that Format 1 constructs work to regress tumors in vivo, using two different tumor cell lines implanted into mice using the protocols in the Examples. Anti-tumor activity with the hemi-COBRA constructs (Pro77 and Pro53) was dependent on the inclusion of both the anti-EGFR sdABDs and the MMP9 cleavable linkers, along with the active anti-CD3 Fv. id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39"
id="p-39"
[0039] Figure 16 shows the schematic of the next generation format, a full length construct that has two pseudo Fv domains with cleavable sites between them, as is generally described in US 2018/0134789, hereby incorporated by reference. However, as shown in the following figures, this first generation full length construct does not show very good conditionality, as it can isomerize to form both an active and inactive construct. id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40"
id="p-40"
[0040] Figure 17 shows that the Format 3A construct pairs actually show better conditionality than the Pro100 first generation full length constructs. id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41"
id="p-41"
[0041] Figure 18 depicts additional first generation full length constructs that were tested in Figure19. id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42"
id="p-42"
[0042] Figure 19 shows that the first generation constructs show high activity even in the uncleaved format, e.g. poor conditionality. id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43"
id="p-43"
[0043] Figure 20 shows that the first generation full length constructs show two monomer peaks on analytical SEC. id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44"
id="p-44"
[0044] Figure 21 shows the schematic of the reason for the noncleaved activity, which is that the full length first generation constructs isomerize to form two conformations, one which is inactive since there is no active anti-CD3 Fv formed (the "bivalent scFv"), and the other which is active in the absence of protease, a "single chain diabody" type of configuration. See PEDS 23(8):667-677 (2010). id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45"
id="p-45"
[0045] Figure 22 shows the results of a TDCC assay, run at 37C for 2 days, with the first generation single chain constructs. The results show that the uncleaved constructs show strong killing. These results led to the generation of the Format 1 constructs. 118459-5005-WO id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46"
id="p-46"
[0046] Figure 23A - Figure 23G shows Format 1 constructs used in the invention. As will be appreciated by those in the art and described herein, these are depicted with a sdABD-EGFR targeting moiety, although sdABDs to other TTAs can be used. id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47"
id="p-47"
[0047] Figure 24 shows that the Format 1 constructs (Pro140 in this case) form a single isomer that is stable at 37C. id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48"
id="p-48"
[0048] Figure 25 depicts that Format 1 constructs have very low binding to human CD3 in the uncleaved format, as measured by an Octet assay. The top line is Pro120, the middle line is Pro51 (the positive control) and the bottom lines are Pro140 held at either 4C or 37C for days. id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49"
id="p-49"
[0049] Figure 26 similarly depicts that the Format 1 constructs have very low TDCC activity in the uncleaved form. id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50"
id="p-50"
[0050] Figure 27 depicts a specific Format 1 construct, Pro140, used in in vivo testing, using sdABD-EGFR as the targeting moieties and an MMP9 cleavage site. id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51"
id="p-51"
[0051] Figure 28A – Figure 28B shows tumor regression using a Format 1 construct. id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52"
id="p-52"
[0052] Figure 29 depicts that due to the cleavage site in the constrained Fv, several different fragments can be generated: a partially cleaved fragment and the fully cleaved fragment. Surprisingly, the partially cleaved format is more active than the fully cleaved format, leading to the generation of Format 2. id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53"
id="p-53"
[0053] Figure 30 shows a number of Format 2 schematics, all of which use sdABD-EGFR targeting domains, although as outlined herein and listed in the sequences, sdABDs to other TTAs can be used. Pro51 and Pro201 are positive controls (in an active "hemi" configuration), and Pro214 is a full length negative control, as there is no cleavage site. id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54"
id="p-54"
[0054] Figure 31 shows the TDCC activity of Format 2 construct Pro187, which uses a meprin cleavage site. Pro187 in the TDCC assay was 1200-fold more active when added pre-cleaved than when added uncleaved. The pre-cleaved Pro187 demonstrated activity that fell between the positive controls Pro51 and Pro201. The uncleaved Pro187 demonstrated activity similar to Pro214, which does not contain a protease cleavable linker. id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55"
id="p-55"
[0055] Figure 32 shows the TDCC activity of Format 2 construct Pro186, which uses n MMP9 cleavage site. Pro186 in the TDCC assay was 18-fold more active when added pre-cleaved than when added uncleaved. The pre-cleaved Pro186 demonstrated activity that fell 118459-5005-WO between the positive controls Pro51 and Pro201. The uncleaved Pro186 demonstrated more activity than Pro214, which does not contain a protease cleavable linker. id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56"
id="p-56"
[0056] Figure 33 depicts that the Pro186 construct binds to cells that have different levels of EGFR receptors, with CHO cells not expressing EGFR on the cell surface. Pro186 saturates cells expressing differing levels of EGFR at similar COBRA concentrations. id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57"
id="p-57"
[0057] Figure 34 shows the schematics of Format 2 constructs used in the in vivo studies of Figure35, all of which use sdABD-EGFR targeting domains. id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58"
id="p-58"
[0058] Figure 35 shows that the Format 2 construct Pro186 is highly efficacious at both concentrations, and better than the Format 1 construct Pro140 at the lower concentration. id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59"
id="p-59"
[0059] Figure 36 depicts a number of Format 2 constructs based on Pro186 but with different protease cleavage sites. While all of these constructs utilize sdABD-EGFRs for both targeting domains, other sdABDs to different TTAs can be used, and can be the same or different. That is, both homo-targeting (both targeting sdABDs to the same TTA) or hetero-targeting (one sdABD to a first TTA and the other to a different TTA) can be done. id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60"
id="p-60"
[0060] Figure 37 depicts the schematics for different Format 2 constructs that vary linker length between the Fv domains. These are shown using an MMP9 cleavage site, although others can be used as outlined herein. Similarly, while all of these constructs utilize sdABD-EGFRs for both targeting domains, other sdABDs to different TTAs can be used, and can be the same or different. id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61"
id="p-61"
[0061] Figure 38 shows that the linker length for the pseudo Fv can be varied, e.g. that a Format 2 construct with a short linker between the active Fv ("short active") with a longer linker between the pseudo Fv ("long inactive") exhibits similar activity to a "short active" with a "short inactive". Thus conditionality of the COBRA construct is not dependent on both the active and inactive scFv linkers being constrained; as long as one of them is constrained, single chain diabody folding appears to be favored over bivalent scFv folding. id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62"
id="p-62"
[0062] Figure 39 shows that the linker length for the active Fv can be varied, e.g. that Format constructs with "long active" and "short inactive" behaves similarly to a "short active" and "short inactive" construct. Thus conditionality of the COBRA construct is not dependent on both the active and inactive scFv linkers being constrained; as long as one of them is constrained, single chain diabody folding appears to be favored over bivalent scFv folding. 118459-5005-WO id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63"
id="p-63"
[0063] Figure 40A – Figure 40C shows the schematics for a number of different constructs. Pro188 is a Format 1 construct which is similar to Pro140 except with a long linker (16mer) in the pseudo Fv. Pro189 and Pro190 (Format 2 constructs) are similar to Pro186 and Pro1except with a long linker (16mer) in the pseudo Fv domain. Pro191 and Pro192 (also Format constructs) are similar to Pro189 and Pro190 except they have an additional cleavage site upstream of the sdABD(1/2). Pro193 (Format 4) has a single EGFR targeting domain, the iVH and iVL rearranged to be in reversed order, and an additional cleavage site upstream of the sdABD(1/2). Pro195 is a Format 2 construct similar to Pro186, with targeting domains that bind to the same TTA, EGFR, but to different epitopes. Pro196, Pro197 and Pro198 are Format 2 constructs with rearranged variable domains. id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64"
id="p-64"
[0064] Figure 41 depicts the fact that different sdABD clones directed to human FOLRshow differential killing. A Pro22 type construct (Pro51 with a FLAG sequence instead of a NCL) that binds to human FOLR1 was compared to a Pro22-EGFR construct against a number of cell line families. id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65"
id="p-65"
[0065] Figure 42 depicts the schematics for four sdABD-FOLR1 constructs, including the use of the Pro201 positive control using sdABD-EGFR2 (with two molecules intermolecularly associating to form two active Fvs against CD3), and two Format 2 test articles, Pro311, using the h77.2 sdABD and Pro312 using the h59.3 sdABD, as well as two negative controls, Pro299, using the h77.2 sdABD and Pro303 using the h59.3 sdABD. id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66"
id="p-66"
[0066] Figure 43 depicts the schematics of the Format 2 constructs for the FOLR/MMP9 in vivo design. id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67"
id="p-67"
[0067] Figure 44 shows the efficacy of the Pro312 construct in vivo, and demonstrates the MMP9 cleavable linker is necessary for anti-tumor activity. id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68"
id="p-68"
[0068] Figure 45 depicts the schematics of some formats using sdABDs to human B7H(sdABD-B7H3), including Pro244, the positive control (using sdABD-B7H3 (hF7) (with two molecules intermolecularly associating to form two active Fvs against CD3), and two Format test articles, Pro225, a Format 2 construct, and Pro295, the negative control lacking a cleavage site. id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69"
id="p-69"
[0069] Figure 46 shows that Pro225 has great conditionality as compared to the control, Pro295. 118459-5005-WO id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70"
id="p-70"
[0070] Figure 47 shows that a Format 2 construct of using a meprin linker, Pro373, shows great conditionality compared to Pro295. id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71"
id="p-71"
[0071] Figure 48 depicts a number of sdABD-B7H3 (using the hF12 sequence) constructs, showing the Pro51 positive control using sdABD-EGFR, the Pro244 positive control using sdABD-hF12 B7H3, the test construct, Pro226, and the negative control Pro296 without a cleavage site. id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72"
id="p-72"
[0072] Figure 49 shows the good conditionality of the Pro226 construct in a TDCC assay. id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73"
id="p-73"
[0073] Figure 50 shows the humanization of sdABDs to human EpCAM. id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74"
id="p-74"
[0074] Figure 51 shows the schematics of a number of Formats: Pro22hVIB13 and Pro2are positive controls, Pro199 is a Format 2 construct and Pro175 is the negative control. id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75"
id="p-75"
[0075] Figure 52 shows the TDCC activity of sdABD-EpCAM constructs, showing good conditionality. id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76"
id="p-76"
[0076] Figure 53A – Figure 53B shows the TDCC activity of sdABD-EpCAM Pro1construct, showing good conditionality in HT29 and LoVo cell models. id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77"
id="p-77"
[0077] Figure 54A – Figure 54B shows the TDCC activity of sdABD-EpCAM Pro2construct, showing good conditionality in HT29 and LoVo cell models. id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78"
id="p-78"
[0078] Figure 55 shows a schematic of Pro255, which uses two different sdABD-TTA, one to EGFR (sdABD-EGFR) and the other to EpCAM (sdABD-EpCAM), as compared to Pro199, with dual EpCAM sdABDs. These are sometimes referred to herein as "hetero-targeting" constructs, in this case, a Format 2 construct. id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79"
id="p-79"
[0079] Figure 56 shows that the Pro255 dual targeting molecule with an MMP9 cleavage site, shows good conditionality. id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80"
id="p-80"
[0080] Figure 57A – Figure 57D shows the results of experiments on three different cell types. First, Raji transfectants were created with similar expression levels of EpCAM, EGFR and EpCAM + EGFR (data not shown). Then Pro255, which targets both EpCAM and EGFR, was tested in TDCC assays using each cell type. Figure 57A shows the parental Raji line, that doesn’t express either receptor. Figure 57B shows conditionality on the EpCAM line. Figure 57C shows conditionality on the EGRF line. Figure 57D shows conditionality on the EpCAM/EGFR line. id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81"
id="p-81"
[0081] Figure 58 depicts the schematics of a Format 4 construct, Pro258. 118459-5005-WO id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82"
id="p-82"
[0082] Figure 59A – Figure 59B shows that Pro258 is conditional in both FBS and human serum. The conditionality of the MMP9 linker is underestimated due to the MMP9 activity in the culture. Interestingly, Pro51 TDCC activity is inhibited by HSA binding while Pro2TDCC activity is similar to Pro51 in the presence of HSA. Finally, the Pro258 conditionality is somewhat enhanced in the presence of HSA by 6X. id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83"
id="p-83"
[0083] Figure 60A – Figure 60C shows the cleavage of the MMP9 substrate by other MMPs. id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84"
id="p-84"
[0084] Figure 61A – Figure 61B shows some of the exemplary constructs and their formats. id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85"
id="p-85"
[0085] Figure 62A – Figure 62U shows a number of sequences of the invention, although many additional sequences are also found in the sequence listing. CDRs are underlined and bolded, linkers are double underlined (with cleavable linkers being italicized and double underlined) and domain separations are indicated by "/". All His6 tags are optional, as they can be used to reduce immunogenicity in humans as well as be purification tags. VI. DETAILED DESCRIPTION OF THE INVENTION A. Introduction id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86"
id="p-86"
[0086] The present invention is directed to methods of reducing the toxicity and side effects of bispecific antibodies (including antibody-like functional proteins) that bind to important physiological targets such as CD3 and tumor antigens. Many antigen binding proteins, such as antibodies, can have significant off-target side effects, and thus there is a need to only activate the binding capabilities of a therapeutic molecule in the vicinity of the disease tissue, to avoid off-target interactions. Accordingly, the present invention is directed to multivalent conditionally effective ("MCE") proteins that have a number of functional protein domains. In general, one of these domains is an antigen binding domain (ABD) that will bind a target tumor antigen (TTA), and another is an ABD that will bind a T-cell antigen such as CD3 under certain conditions. Additionally, the MCE proteins also include one or more protease cleavage sites. That is, the therapeutic molecules are made in a "pro-drug" like format, wherein the CD3 binding domain is inactive until exposed to a tumor environment. The tumor environment contains proteases, such that upon exposure to the protease, the prodrug is cleaved and becomes active. 118459-5005-WO id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87"
id="p-87"
[0087] This is generally accomplished herein by using proteins that include a "pseudo" variable heavy domain and a "pseudo" variable light domain directed to the T-cell antigen such as CD3, that restrain the CD3 Fvs of the MCE into an inactive format as is discussed herein. As the TTA targets the MCE into the proximity of the tumor, the MCE is thus exposed to the protease. Upon cleavage, the active variable heavy domain and active light domain are now able to pair to form one or more active ABDs to CD3 and thus recruit T cells to the tumor, resulting in treatment. id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88"
id="p-88"
[0088] In general, the CD3 binding domain ("Fv") is in a constrained format, wherein the linker between the active variable heavy domain and the active variable light domain that traditionally form an Fv is too short to allow the two active variable domains to bind each other; this is referred to as "constrained linker"; these can be constrained and cleavable (CCL, as used in Format 1) or constrained and not cleavable (CNCL, as used in Format 2). Rather, in the prodrug (e.g. uncleaved) format, the prodrug polypeptide also comprises a "pseudo Fv domain". The pseudo Fv domain comprises a variable heavy and light domain, with standard framework regions, but "inert" or "inactive" CDRs. The pseudo Fv domain also has a constrained linker between the inactive variable heavy and inactive variable light domains. Since neither Fv nor pseudo Fv domains can self-assemble due to the steric constraints, there is an intramolecular assembly that pairs the aVL with the iVH and the aVH with the iVL, due to the affinity of the framework regions of each. However, due to the "inert" CDRs of the pseudo domain, the resulting ABDs will not bind CD3, thus preventing off target toxicities. However, in the presence of proteases that are in or near the tumor, the prodrug construct is cleaved such that the pseudo-Fv domain is released from the surface and thus allows the "real" variable heavy and variable light domains to associate intermolecularly (e.g. two cleaved constructs come together), thus triggering active CDbinding and the resulting tumor efficacy. These constructs are generally referred to herein as CO nditional B ispecific R edirected A ctivation constructs, or "COBRAs™". The stability of the intramolecular assembly is shown by the conditionality experiments herein, whereby in the absence of protease, the uncleaved constructs have no activity (e.g. no active CDbinding domain is formed). 118459-5005-WO id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89"
id="p-89"
[0089] Interestingly, for ease of description, while these constructs are all referred to herein as "constrained", additional work shows that the intramolecular assembly is favored even if one of the Fv domains is not constrained, e.g. one of the domains can have a longer, flexible linker. That is, as shown in Figure , Figure and Figure, intramolecular assembly still occurs (e.g. the uncleaved constructs are inactive in the absence of protease cleavage) if only one of the Fv domains, either the one with an active VL and VH, or the pseudo Fv domain, is constrained. However, in the current systems, when both linkers are constrained, the protein has better expression. However, as will be appreciated by those of skill in the art, any of the Format 1, Format 2 or Format 4 constructs herein can have one of these Fv domains with an "unconstrained" or "flexible" linker. For ease of reference, the constructs are shown with both Fv domains in a constrained format. id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90"
id="p-90"
[0090] The constructs and formats of the invention are variations over inventions described in WO2017/156178, hereby expressly incorporated by reference in its entirety. As shown in Figure, previous constructs have the ability to isomerize due to the presence of two sets of VH and VL domains in a single polypeptide, forming both a bivalent scFv and a single chain diabody. Even after purification of each isoform, the bivalent construct can still reach equilibrium with the diabody isoform. As the single chain diabody has the ability to bind to CD3 in the absence of protease cleavage, the utility of the construct is diminished. id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91"
id="p-91"
[0091] To solve this issue, the present invention provides for four separate types of constructs to accomplish this conditional activation. The prodrug activation can happen in one of four general ways, as is generally shown in the Figures. In Figure 1, a "format 1" mechanism is shown. In this embodiment, the prodrug construct has two cleavage sites: one between the VH and vl domains of the constrained Fv, thus freeing the two variable domains to associate, and a second at a site that releases the pseudo Fv domain from the prodrug construct, leaving two molecules that associate due to the innate self-assembly of the variable heavy and variable light domains, each having an antigen binding domain to a tumor antigen as well, thus allowing the recruitment of T cells to the tumor site. id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92"
id="p-92"
[0092] In an alternate embodiment, the prodrug construct is shown in Figure 2, a "format 2" mechanism. In this embodiment, the domain linker between the active variable heavy and active light chains is a constrained but not cleavable linker ("CNCL"). In the prodrug 118459-5005-WO format, the inactive VH and VL of the constrained pseudo Fv domain associate with the VH and VL of the constrained Fv domain, such that there is no CD3 binding. However, once cleavage in the tumor environment happens, two different activated proteins, each comprising an active variable heavy and light domain, associate to form two anti-CDbinding domains. id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93"
id="p-93"
[0093] In addition to the "single chain protein" COBRA formats discussed above, where all of the components are contained on a single amino acid sequence, there are also constructs that rely on two proteins "hemi-COBRAs", which act in pairs, as shown in Figure 3. In this embodiment, each protein has one active and one inert variable domain separated by a protease cleavage site. Each molecule contains a TTA binding domain, such that when the molecules are bound to the TTA and exposed to tumor protease, the inert domains are cleaved off and the two active variable domains self-assemble to form an anti-CD3 binding domain. id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94"
id="p-94"
[0094] Furthermore, the invention provides "format 4" constructs as well, as depicted in Figure 4. These are similar to the "format 2" designs, except that a single ABD to a TTA is used, such that upon cleavage, two of the pro-drug molecules now form a tetravalent, bispecific construct containing two active anti-CD3 domains, as is further described below. id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95"
id="p-95"
[0095] Accordingly, the formats and constructs of the invention find use in the treatment of disease. B. Definitions id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96"
id="p-96"
[0096] In order that the application may be more completely understood, several definitions are set forth below. Such definitions are meant to encompass grammatical equivalents. id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97"
id="p-97"
[0097] By "amino acid" and "amino acid identity" as used herein is meant one of the naturally occurring amino acids or any non-natural analogues that may be present at a specific, defined position. In many embodiments, "amino acid" means one of the naturally occurring amino acids. By "protein" herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98"
id="p-98"
[0098] By "amino acid modification" herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence or an alteration to a moiety chemically linked to a 118459-5005-WO protein. For example, a modification may be an altered carbohydrate or PEG structure attached to a protein. For clarity, unless otherwise noted, the amino acid modification is always to an amino acid coded for by DNA, e.g. the 20 amino acids that have codons in DNA and RNA. The preferred amino acid modification herein is a substitution. id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99"
id="p-99"
[0099] By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is to an amino acid that is not naturally occurring at the particular position, either not naturally occurring within the organism or in any organism. For clarity, a protein which has been engineered to change the nucleic acid coding sequence but not change the starting amino acid (for example exchanging CGG (encoding arginine) to CGA (still encoding arginine) to increase host organism expression levels) is not an "amino acid substitution"; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the particular position that it started with, it is not an amino acid substitution. id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100"
id="p-100"
[00100] By "amino acid insertion" or "insertion" as used herein is meant the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101"
id="p-101"
[00101] By "amino acid deletion" or "deletion" as used herein is meant the removal of an amino acid sequence at a particular position in a parent polypeptide sequence. id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102" id="p-102"
id="p-102"
[00102] The polypeptides of the invention specifically bind to CD3 and target tumor antigens (TTAs) such as target cell receptors, as outlined herein. "Specific binding" or "specifically binds to" or is "specific for" a particular antigen or an epitope means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target. id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103"
id="p-103"
[00103] Specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KD for an antigen or epitope of at least about 10-4 M, at least about 10-5 M, at least about 10-6 M, at least about 10-7 M, at least about 10-8 M, at least 118459-5005-WO about 10-9 M, alternatively at least about 10-10 M, at least about 10-11 M, at least about 10-12 M, or greater, where KD refers to a dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the antigen or epitope. id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104" id="p-104"
id="p-104"
[00104] Also, specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KA or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction. Binding affinity is generally measured using a Biacore assay or Octet as is known in the art. id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105" id="p-105"
id="p-105"
[00105] By "parent polypeptide" or "precursor polypeptide" (including Fc parent or precursors) as used herein is meant a polypeptide that is subsequently modified to generate a variant. Said parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide. Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, or the amino acid sequence that encodes it. Accordingly, by "parent Fc polypeptide" as used herein is meant an unmodified Fc polypeptide that is modified to generate a variant, and by "parent antibody" as used herein is meant an unmodified antibody that is modified to generate a variant antibody. id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106" id="p-106"
id="p-106"
[00106] By "position" as used herein is meant a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example the EU index for antibody numbering. id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107" id="p-107"
id="p-107"
[00107] By "target antigen" as used herein is meant the molecule that is bound specifically by the variable region of a given antibody. A target antigen may be a protein, carbohydrate, lipid, or other chemical compound. A range of suitable exemplary target antigens are described herein. id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108" id="p-108"
id="p-108"
[00108] By "target cell" as used herein is meant a cell that expresses a target antigen. Generally, for the purposes of the invention, target cells are either tumor cells that express TTAs or T cells that express the CD3 antigen. 118459-5005-WO id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109"
id="p-109"
[00109] By "Fv" or "Fv domain" or "Fv region" as used herein is meant a polypeptide that comprises the VL and VH domains of an antigen binding domain, generally from an antibody. Fv domains usually form an "antigen binding domain" or "ABD" as discussed herein, if they contain active VH and VL domains (although in some cases, an Fv containing a constrained linker is used, such that an active ABD isn't formed prior to cleavage). As discussed below, Fv domains can be organized in a number of ways in the present invention, and can be "active" or "inactive", such as in a scFv format, a constrained Fv format, a pseudo Fv format, etc. It should be understood that in the present invention, in some cases an Fv domain is made up of a VH and VL domain on a single polypeptide chain, such as shown in Figure 1 and Figure 2 but with a constrained linker such that an intramolecular ABD cannot be formed. In these embodiments, it is after cleavage that two active ABDs are formed. In some cases an Fv domain is made up of a VH and a VL domain, one of which is inert, such that only after cleavage is an intermolecular ABD formed. As discussed below, Fv domains can be organized in a number of ways in the present invention, and can be "active" or "inactive", such as in a scFv format, a constrained Fv format, a pseudo Fv format, etc. In addition, as discussed herein, Fv domains containing VH and VL can be/form ABDs, and other ABDs that do not contain VH and VL domains can be formed using sdABDs. id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110" id="p-110"
id="p-110"
[00110] By "variable domain" herein is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the Vκ, Vλ, and/or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively. In some cases, a single variable domain, such as a sdFv (also referred to herein as sdABD) can be used. id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111" id="p-111"
id="p-111"
[00111] In embodiments utilizing both variable heavy (VH) and variable light (VL) domains, each VH and VL is composed of three hypervariable regions ("complementary determining regions," "CDRs") and four "framework regions", or "FRs", arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, the VH domain has the structure vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4 and the VL domain has the structure vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4. As is more fully described herein, the vhFR regions and the vlFR regions 118459-5005-WO self assemble to form Fv domains. In general, in the prodrug formats of the invention, there are "constrained Fv domains" wherein the VH and VL domains cannot self associate, and "pseudo Fv domains" for which the CDRs do not form antigen binding domains when self associated. id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112"
id="p-112"
[00112] The hypervariable regions confer antigen binding specificity and generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1; "L" denotes light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and around about 31-35B (HCDR1; "H" denotes heavy chain), 50-65 (HCDR2), and 95-1(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or those residues forming a hypervariable loop (e.g. residues 26-(LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-(HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Specific CDRs of the invention are described below. id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113" id="p-113"
id="p-113"
[00113] As will be appreciated by those in the art, the exact numbering and placement of the CDRs can be different among different numbering systems. However, it should be understood that the disclosure of a variable heavy and/or variable light sequence includes the disclosure of the associated (inherent) CDRs. Accordingly, the disclosure of each variable heavy region is a disclosure of the vhCDRs (e.g. vhCDR1, vhCDR2 and vhCDR3) and the disclosure of each variable light region is a disclosure of the vlCDRs (e.g. vlCDR1, vlCDR2 and vlCDR3). id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114"
id="p-114"
[00114] A useful comparison of CDR numbering is as below, see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003):
Claims (49)
1./ CLAIMS: 1. A polypeptide comprising, from N- to C-terminal: a) a first single domain antigen binding domain (sdABD) that binds to a human tumor target antigen (TTA) (sdABD-TTA); b) a first domain linker; c) a constrained Fv domain comprising: i) a first variable heavy domain comprising a vhCDR1, vhCDR2 and vhCDR3; ii) a constrained non-cleavable linker (CNCL); and iii) a first variable light domain comprising vlCDR1, vlCDR2 and vlCDR3; wherein the CNCL is positioned between the variable heavy domain and the variable light domain and prevents said variable heavy domain and said variable light domain from interacting to form an active Fv capable of binding CD3; d) a second domain linker; e) a second sdABD-TTA; f) a cleavable linker (CL); g) a pseudo Fv domain comprising: i) a first pseudo variable light domain; ii) a non-cleavable linker (NCL); and iii) a first pseudo variable heavy domain; h) a third domain linker; and i) a third sdABD that binds to human serum albumin; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 but said constrained Fv domain does not bind CD3; and the polypeptide does not bind CD3 when the CL is intact.
2. A polypeptide comprising, from N- to C-terminal: a) a first sdABD-TTA; b) a first domain linker; c) a constrained Fv domain comprising: i) a first variable heavy domain comprising a vhCDR1, vhCDR2 and vhCDR3; ii) a constrained cleavable linker (CCL); and iii) a first variable light domain comprising vlCDR1, vlCDR2 and vlCDR3; 273119/ d) a second domain linker; e) a second sdABD-TTA; f) a cleavable linker (CL); g) a pseudo Fv domain comprising: i) a first pseudo variable heavy domain; ii) a non-cleavable linker (NCL); and iii) a first pseudo variable light domain; h) a third domain linker; and i) a third sdABD that binds to human serum albumin; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 but said constrained Fv domain does not bind CD3; and the polypeptide does not bind CD3 when the CL is intact.
3. A polypeptide according to any one of claims 1 to 2 wherein said first variable heavy domain is N-terminal to said first variable light domain and said pseudo variable light domain is N-terminal to said pseudo variable heavy domain.
4. A polypeptide according to any one of claims 1 to 2 wherein said first variable heavy domain is N-terminal to said first variable light domain and said pseudo variable heavy domain is N-terminal to said pseudo variable light domain.
5. A polypeptide according to any one of claims 1 to 2 wherein said first variable light domain is N-terminal to said first variable heavy domain and said pseudo variable light domain is N-terminal to said pseudo variable heavy domain.
6. A polypeptide according to any one of claims 1 to 2 wherein said first variable light domain is N-terminal to said first variable heavy domain and said pseudo variable heavy domain is N-terminal to said pseudo variable light domain.
7. A polypeptide according to any one of claims 1-6 wherein said first and second TTA is the same.
8. A polypeptide according to any one of claims 1-6 wherein said first and second TTA are different.
9. A polypeptide according to any one of claims 1-8 wherein said first and second TTA are selected from EGFR, EpCAM, FOLR1 and B7H3. 273119/
10. A polypeptide according to any one of claims 1-9 wherein said first and second sdABD-TTAs are selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29; SEQ ID NO:33; SEQ ID NO:37 and SEQ ID NO:41.
11. A polypeptide according to any one claim 1 to 10 wherein said half-life extension domain has SEQ ID NO:45.
12. A polypeptide according to any one claim 1 to 11 wherein said cleavable linker is cleaved by a human protease selected from the group consisting of MMP2, MMP9, Meprin A, Meprin B, Cathepsin S, Cathepsin K, Cathepsin L, GranzymeB, uPA, Kallikrein 7, matriptase and thrombin.
13. A polypeptide according to claim 1 comprising a protein selected from the group consisting of Pro226, Pro233, Pro311, Pro312, Pro313, Pro495, Pro246, Pro254, Pro255, Pro256, Pro420, Pro421, Pro432, Pro479, Pro480, Pro187, Pro221, Pro222, Pro223, Pro224, Pro393, Pro394, Pro395, Pro396, Pro429, Pro430 and Pro431.
14. A nucleic acid encoding a polypeptide according to any one of claims 1 to 13.
15. An expression vector comprising the nucleic acid of claim 14.
16. A host cell comprising the expression vector of claim 15.
17. A method of making a polypeptide comprising culturing the host cell of claim 16 under conditions wherein said polypeptide is expressed and recovering said polypeptide.
18. The polypeptide of any one of claims 1 to 13 for use in a method of treating cancer comprising administering said polypeptide to a patient.
19. A composition comprising: a) a first polypeptide comprising, from N- to C-terminal: i) a first sdABD that binds a human tumor target antigen; ii) a first domain linker; iii) a first pseudo Fv domain comprising, from N- to C-terminal: 1) a variable heavy chain comprising a vhCDR1, vhCDR2 and vhCDR3; 2) a first cleavable linker; and 3) a first pseudo variable light domain comprising iVLCDR1, iVLCDR2 and iVLCDR3; 273119/ iv) a second domain linker; v) a second sdABD that binds to human serum albumin; b) a second polypeptide comprising, from N- to C-terminal: i) a third sdABD that binds to a human tumor target antigen; ii) a third domain linker; iii) a second pseudo Fv domain comprising, from N- to C-terminal: 1) a variable light chain comprising a VLCDR1, VLCDR2 and VLCDR3; 2) a second cleavable linker; and 3) a first pseudo variable heavy domain comprising iVHCDR1, iVHCDR2 and iVHCDR3; iv) a fourth domain linker; v) a fourth sdABD that binds to human serum albumin; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 when associated; said first variable heavy domain and said first pseudo variable light domain intermolecularly associate to form an inactive Fv; said first variable light domain and said first pseudo variable heavy domain intermolecularly associate to form an inactive Fv; wherein said first and third sdABD are selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29; SEQ ID NO:33; SEQ ID NO:37 and SEQ ID NO:41.
20. A composition according to claim 19 wherein said first sdABD from the first polypeptide and the third sdABD from the second polypeptide target the same TTA.
21. A composition according to claim 19 wherein said first sdABD from the first polypeptide and the third sdABD from the second polypeptide target different TTAs.
22. A composition according to any one of claims 19 to 21 wherein said second and fourth sdABDs have SEQ ID NO:45.
23. A nucleic acid composition comprising: 273119/ a) a first nucleic acid encoding a first polypeptide according to the composition of any one of claims 19 to 22; and b) a second nucleic acid encoding a second polypeptide according to the composition of any one of claims 19 to 22.
24. An expression vector composition comprising: a) a first expression vector comprising the first nucleic acid of claim 23; and b) a second expression vector comprising the second nucleic acid of claim 23.
25. A host cell comprising the expression vector composition of claim 24.
26. A method of making a composition comprising culturing the host cell of claim 25 under conditions wherein said polypeptides are expressed and recovering said polypeptides.
27. The composition of any one of claims 19 to 22 for use in a method of treating cancer comprising administering said composition to a patient.
28. A composition comprising: a) a first polypeptide comprising, from N- to C-terminal: i) a first sdABD that binds a human tumor target antigen; ii) a first domain linker; iii) a second sdABD that binds a human tumor target antigen; iv) a second domain linker; v) a first pseudo Fv domain comprising, from N- to C-terminal: 1) a variable heavy chain comprising a vhCDR1, vhCDR2 and vhCDR3; 2) a first cleavable linker; and 3) a first pseudo variable light domain comprising iVLCDR1, iVLCDR2 and iVLCDR3; vi) a third domain linker; and vii) a third sdABD that binds human serum albumin; b) a second polypeptide comprising, from N- to C-terminal: i) a fourth sdABD that binds a human tumor target antigen; ii) a fourth domain linker; iii) a fifth sdABD that binds a human tumor target antigen; iv) a fifth domain linker; 273119/ v) a second pseudo Fv domain comprising, from N- to C-terminal: 1) a variable light chain comprising a VLCDR1, VLCDR2 and VLCDR3; 2) a second cleavable linker; and 3) a first pseudo variable heavy domain comprising iVHCDR1, iVHCDR2 and iVHCDR3; vi) a sixth domain linker; vii) a sixth sdABD that binds human serum albumin; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 when associated; said first variable heavy domain and said first pseudo variable light domain intermolecularly associate to form an inactive Fv; said first variable light domain and said first pseudo variable heavy domain intermolecularly associate to form an inactive Fv.
29. A composition according to claim 28 wherein said sdABD-HSAs have SEQ ID NO:45.
30. A nucleic acid composition comprising: a) a first nucleic acid encoding a first polypeptide according to the composition of any one of claims 28 to 29; and b) a second nucleic acid encoding a second polypeptide according to the composition of any one of claims 28 to 29, respectively.
31. An expression vector composition comprising: a) a first expression vector comprising the first nucleic acid of claim 30; and b) a second expression vector comprising the second nucleic acid of claim 30.
32. A host cell comprising the expression vector composition of claim 31.
33. A method of making a composition comprising culturing the host cell of claim 32 under conditions wherein said polypeptides are expressed and recovering said polypeptides.
34. The composition of any one of claims 28 to 29 for use in a method of treating cancer comprising administering said composition to a patient.
35. A polypeptide comprising, from N- to C-terminal: a) a first sdABD that binds to a human tumor target antigen (TTA) (sdABD-TTA); 273119/ b) a first domain linker; c) a constrained Fv domain comprising: i) a first variable heavy domain comprising a vhCDR1, vhCDR2 and vhCDR3; ii) a constrained non-cleavable linker (CNCL); and iii) a first variable light domain comprising vlCDR1, vlCDR2 and vlCDR3; d) a first cleavable linker (CL); e) a second sdABD that binds to human serum albumin; f) a second domain linker; g) a pseudo Fv domain comprising: i) a first pseudo variable light domain; ii) a non-cleavable linker (NCL); and iii) a first pseudo variable heavy domain; wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 but said constrained Fv domain does not bind CD3; said first variable heavy domain and said first pseudo variable light domain intermolecularly associate to form an inactive Fv; and said first variable light domain and said first pseudo variable heavy domain from the pseudo Fv intramolecularly associate to form an inactive Fv.
36. A polypeptide according to claim 35 wherein said first variable heavy domain is N-terminal to said first variable light domain and said pseudo variable light domain is N-terminal to said pseudo variable heavy domain.
37. A polypeptide according to claim 35 wherein said first variable heavy domain is N-terminal to said first variable light domain and said pseudo variable heavy domain is N-terminal to said pseudo variable light domain.
38. A polypeptide according to claim 35 wherein said first variable light domain is N-terminal to said first variable heavy domain and said pseudo variable light domain is N-terminal to said pseudo variable heavy domain.
39. A polypeptide according to claim 35 wherein said first variable light domain is N-terminal to said first variable heavy domain and said pseudo variable heavy domain is N-terminal to said pseudo variable light domain. 273119/
40. A polypeptide according to claim 35 to 39 wherein said TTA is selected from EGFR, EpCAM, FOLR1 and B7H3.
41. A polypeptide according to any one of claims 35 to 40 wherein said sdABD-TTA is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29; SEQ ID NO:33; SEQ ID NO:37 and SEQ ID NO:41.
42. A polypeptide according to any one of claims 35 to 41 wherein said half-life extension domain has SEQ ID NO:45.
43. A polypeptide according to any one of claims 35 to 42 wherein said cleavable linkers are cleaved by a human protease selected from the group consisting of of MMP2, MMP9, Meprin A, Meprin B, Cathepsin S, Cathepsin K, Cathepsin L, GranzymeB, uPA, Kallikrein 7, matriptase and thrombin.
44. A polypeptide according to claim 35 comprising Pro258, Pro356, Pro359, Pro388 and Pro364.
45. A nucleic acid encoding a polypeptide according to any one of claims 35 to 44.
46. An expression vector comprising the nucleic acid of claim 45.
47. A host cell comprising the expression vector of claim 46.
48. A method of making a polypeptide comprising culturing the host cell of claim 47 under conditions wherein said polypeptide is expressed and recovering said polypeptide.
49. The polypeptide of any one of claims 35 to 44 for use in a method of treating cancer comprising administering said polypeptide to a patient.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201762555943P | 2017-09-08 | 2017-09-08 | |
| US201762586627P | 2017-11-15 | 2017-11-15 | |
| US201762587318P | 2017-11-16 | 2017-11-16 | |
| PCT/US2018/049774 WO2019051102A2 (en) | 2017-09-08 | 2018-09-06 | Constrained conditionally activated binding proteins |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| IL273119A IL273119A (en) | 2020-04-30 |
| IL273119B1 true IL273119B1 (en) | 2023-06-01 |
| IL273119B2 IL273119B2 (en) | 2023-10-01 |
Family
ID=63788010
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IL302613A IL302613A (en) | 2017-09-08 | 2018-09-06 | Constrained conditionally activated binding proteins |
| IL273119A IL273119B2 (en) | 2017-09-08 | 2018-09-06 | Constrained conditionally activated binding proteins |
| IL302614A IL302614A (en) | 2017-09-08 | 2018-09-06 | Constrained conditionally activated binding proteins |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IL302613A IL302613A (en) | 2017-09-08 | 2018-09-06 | Constrained conditionally activated binding proteins |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IL302614A IL302614A (en) | 2017-09-08 | 2018-09-06 | Constrained conditionally activated binding proteins |
Country Status (19)
| Country | Link |
|---|---|
| US (4) | US11406710B2 (en) |
| EP (1) | EP3679067A2 (en) |
| JP (3) | JP7268005B2 (en) |
| KR (1) | KR20200047687A (en) |
| CN (1) | CN111356700A (en) |
| AU (3) | AU2018328291B2 (en) |
| BR (1) | BR112020004543A2 (en) |
| CA (1) | CA3075034A1 (en) |
| CL (3) | CL2020000570A1 (en) |
| EC (1) | ECSP20021323A (en) |
| IL (3) | IL302613A (en) |
| MX (1) | MX2020002667A (en) |
| MY (1) | MY201723A (en) |
| PE (1) | PE20212205A1 (en) |
| PH (1) | PH12020500474A1 (en) |
| SG (1) | SG11202002089RA (en) |
| TW (3) | TW202426489A (en) |
| WO (1) | WO2019051102A2 (en) |
| ZA (1) | ZA202002325B (en) |
Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP7268005B2 (en) | 2017-09-08 | 2023-05-02 | 武田薬品工業株式会社 | Constrained, Conditionally Activated Binding Proteins |
| MX2020014290A (en) | 2018-06-22 | 2021-08-05 | Cugene Inc | Cytokine-based bioactivatable drugs and methods of uses thereof. |
| EP3833444A1 (en) | 2018-08-09 | 2021-06-16 | Maverick Therapeutics, Inc. | Coexpression and purification method of conditionally activated binding proteins |
| JP2022524338A (en) * | 2019-03-05 | 2022-05-02 | 武田薬品工業株式会社 | Restrained and conditionally activated binding protein |
| WO2020181145A1 (en) * | 2019-03-05 | 2020-09-10 | Maverick Therapeutics, Inc. | Conditionally activated binding proteins containing fc regions and moieties targeting tumor antigens |
| WO2020232305A1 (en) * | 2019-05-14 | 2020-11-19 | Werewolf Therapeutics, Inc. | Separation moieties and methods and use thereof |
| WO2021146590A2 (en) * | 2020-01-17 | 2021-07-22 | Aetio Biotherapy, Inc. | Pro-antibody that reduces off-target toxicity |
| EP4090686A4 (en) * | 2020-01-17 | 2024-03-27 | Immune Targeting Inc. | Pro-antibody that reduces off-target toxicity |
| CN115968378A (en) * | 2020-06-03 | 2023-04-14 | 明尼苏达大学董事会 | B7H 3-targeting proteins and methods of use thereof |
| WO2022013225A1 (en) * | 2020-07-13 | 2022-01-20 | Precirix N.V. | Antibody fragment against folr1 |
| CN116419925A (en) | 2020-08-17 | 2023-07-11 | 武田药品工业有限公司 | Binding proteins that are activated by restricted conditions |
| US20230312715A1 (en) * | 2020-09-04 | 2023-10-05 | Takeda Pharmaceutical Company Limited | Constrained conditionally activated binding protein constructs with human serum albumin domains |
| AU2021396172A1 (en) | 2020-12-09 | 2023-07-06 | Janux Therapeutics, Inc. | Compositions and methods related to tumor activated antibodies targeting psma and effector cell antigens |
| AU2021402183A1 (en) | 2020-12-14 | 2023-07-06 | Takeda Pharmaceutical Company Limited | Conditionally bispecific binding proteins |
| WO2022212668A1 (en) * | 2021-03-31 | 2022-10-06 | Heat Biologics, Inc. | Methods and compositions related to tnfrsf25/dr3 agonists |
| BR112023020437A2 (en) * | 2021-04-06 | 2024-04-30 | Takeda Pharmaceuticals Co | THERAPEUTIC METHODS USING CONDITIONALLY RESTRICTED ACTIVATED BINDING PROTEINS |
| AR128593A1 (en) * | 2022-02-23 | 2024-05-22 | Takeda Pharmaceuticals Co | CONDITIONALLY BISPECIFIC BINDING PROTEINS |
| WO2023212662A2 (en) * | 2022-04-28 | 2023-11-02 | Oregon Health & Science University | Compositions and methods for modulating antigen binding activity |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007042289A2 (en) * | 2005-10-11 | 2007-04-19 | Ablynx N.V. | Nanobodies™ and polypeptides against egfr and igf-ir |
| WO2011033105A1 (en) * | 2009-09-18 | 2011-03-24 | Micromet Ag | Dosage regimen for administering an epcamxcd3 bispecific antibody |
| WO2013083809A1 (en) * | 2011-12-09 | 2013-06-13 | Amgen Research (Munich) Gmbh | Prevention of adverse effects caused by epcamxcd3 bispecific antibodies |
| WO2016046778A2 (en) * | 2014-09-25 | 2016-03-31 | Amgen Inc | Protease-activatable bispecific proteins |
| WO2016156570A1 (en) * | 2015-04-02 | 2016-10-06 | Ablynx N.V. | Bispecific cxcr4-cd-4 polypeptides with potent anti-hiv activity |
| WO2016180969A1 (en) * | 2015-05-13 | 2016-11-17 | Ablynx N.V. | T cell recruiting polypeptides based on tcr alpha/beta reactivity |
Family Cites Families (83)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
| US5585362A (en) | 1989-08-22 | 1996-12-17 | The Regents Of The University Of Michigan | Adenovirus vectors for gene therapy |
| US5959177A (en) | 1989-10-27 | 1999-09-28 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
| US7018809B1 (en) | 1991-09-19 | 2006-03-28 | Genentech, Inc. | Expression of functional antibody fragments |
| US5252479A (en) | 1991-11-08 | 1993-10-12 | Research Corporation Technologies, Inc. | Safe vector for gene therapy |
| FR2688514A1 (en) | 1992-03-16 | 1993-09-17 | Centre Nat Rech Scient | Defective recombinant adenoviruses expressing cytokines and antitumour drugs containing them |
| AU680459B2 (en) | 1992-12-03 | 1997-07-31 | Genzyme Corporation | Gene therapy for cystic fibrosis |
| HU223733B1 (en) | 1993-10-25 | 2004-12-28 | Canji, Inc. | Rekombinant adenoviral vector and methods of use |
| US5789199A (en) | 1994-11-03 | 1998-08-04 | Genentech, Inc. | Process for bacterial production of polypeptides |
| US5840523A (en) | 1995-03-01 | 1998-11-24 | Genetech, Inc. | Methods and compositions for secretion of heterologous polypeptides |
| US5731168A (en) | 1995-03-01 | 1998-03-24 | Genentech, Inc. | Method for making heteromultimeric polypeptides |
| US6040498A (en) | 1998-08-11 | 2000-03-21 | North Caroline State University | Genetically engineered duckweed |
| CZ302070B6 (en) | 1998-04-21 | 2010-09-29 | Micromet Ag | Single-chain multifunctional polypeptide, polynucleotide, vector containing this polynucleotide, cell transformed with this polynucleotide, agent containing this polypeptide, polynucleotide or vector and their use as well as method for identification |
| US7125978B1 (en) | 1999-10-04 | 2006-10-24 | Medicago Inc. | Promoter for regulating expression of foreign genes |
| NZ517906A (en) | 1999-10-04 | 2003-01-31 | Medicago Inc | Cloning of genomic sequences encoding nitrite reductase (NiR) for use in regulated expression of foreign genes in host plants |
| JP2004523205A (en) | 2000-07-25 | 2004-08-05 | イムノメディクス， インコーポレイテッド | Multivalent target binding protein |
| EP1354600A1 (en) | 2002-04-19 | 2003-10-22 | Affimed Therapeutics AG | Antibody combination useful for tumor therapy |
| DE10231109A1 (en) | 2002-07-10 | 2004-01-22 | Daimlerchrysler Ag | exhaust turbine |
| AU2003265866A1 (en) | 2002-09-03 | 2004-03-29 | Vit Lauermann | Targeted release |
| US20100003253A1 (en) | 2002-11-08 | 2010-01-07 | Ablynx N.V. | Single domain antibodies directed against epidermal growth factor receptor and uses therefor |
| EP1629012B1 (en) | 2003-05-31 | 2018-11-28 | Amgen Research (Munich) GmbH | Pharmaceutical compositions comprising bispecific anti-cd3, anti-cd19 antibody constructs for the treatment of b-cell related disorders |
| AU2004283850C1 (en) | 2003-10-16 | 2011-11-03 | Amgen Research (Munich) Gmbh | Multispecific deimmunized CD3-binders |
| US20090252681A1 (en) | 2005-10-11 | 2009-10-08 | Ablynx N.V. | Nanobodies and Polypeptides Against EGFR and IGF-IR |
| AU2006327175A1 (en) | 2005-12-21 | 2007-06-28 | Medimmune, Llc | Epha2 bite molecules and uses thereof |
| US20070269422A1 (en) | 2006-05-17 | 2007-11-22 | Ablynx N.V. | Serum albumin binding proteins with long half-lives |
| WO2008119567A2 (en) | 2007-04-03 | 2008-10-09 | Micromet Ag | Cross-species-specific cd3-epsilon binding domain |
| WO2009021754A2 (en) * | 2007-08-15 | 2009-02-19 | Bayer Schering Pharma Aktiengesellschaft | Monospecific and multispecific antibodies and method of use |
| CN106995495A (en) * | 2009-01-12 | 2017-08-01 | 希托马克斯医疗有限责任公司 | Modified antibodies composition and its preparation and application |
| RU2011148913A (en) | 2009-05-01 | 2013-06-10 | Эбботт Лэборетриз | IMMUNOGLOBULIN WITH TWO VARIABLE DOMAINS AND ITS APPLICATION |
| US20110029476A1 (en) | 2009-07-29 | 2011-02-03 | Kas Kasravi | Indicating relationships among text documents including a patent based on characteristics of the text documents |
| US9932403B2 (en) * | 2010-05-20 | 2018-04-03 | Ablynx Nv | Biological materials related to HER3 |
| US9193791B2 (en) | 2010-08-03 | 2015-11-24 | City Of Hope | Development of masked therapeutic antibodies to limit off-target effects |
| WO2012025525A1 (en) | 2010-08-24 | 2012-03-01 | Roche Glycart Ag | Activatable bispecific antibodies |
| US20120225081A1 (en) | 2010-09-03 | 2012-09-06 | Boehringer Ingelheim International Gmbh | Vegf-binding molecules |
| CN103561771B (en) | 2011-03-17 | 2019-01-04 | 伯明翰大学 | The immunization therapy redirected |
| JP6400470B2 (en) | 2011-05-16 | 2018-10-03 | ジェネロン（シャンハイ）コーポレイション リミテッド | Multispecific Fab fusion proteins and methods of use |
| RS58765B1 (en) | 2011-05-21 | 2019-06-28 | Macrogenics Inc | Cd3-binding molecules capable of binding to human and non-human cd3 |
| MX2014000031A (en) | 2011-07-01 | 2014-07-09 | Bayer Ip Gmbh | Relaxin fusion polypeptides and uses thereof. |
| UA118833C2 (en) | 2011-08-17 | 2019-03-25 | Ґлаксо Ґруп Лімітед | Modified proteins and peptides |
| EP3333190A1 (en) | 2012-01-13 | 2018-06-13 | Julius-Maximilians-Universität Würzburg | Dual antigen-induced bipartite functional complementation |
| AP2014007791A0 (en) | 2012-02-27 | 2014-07-31 | Boehringer Ingelheim Int | CX3CR1-binding polypeptides |
| GB201203442D0 (en) | 2012-02-28 | 2012-04-11 | Univ Birmingham | Immunotherapeutic molecules and uses |
| JP6407726B2 (en) | 2012-03-01 | 2018-10-24 | アムゲン リサーチ （ミュンヘン） ゲーエムベーハーＡＭＧＥＮ Ｒｅｓｅａｒｃｈ（Ｍｕｎｉｃｈ）ＧｍｂＨ | Long-life polypeptide binding molecule |
| SG11201406943XA (en) | 2012-04-27 | 2014-12-30 | Cytomx Therapeutics Inc | Activatable antibodies that bind epidermal growth factor receptor and methods of use thereof |
| PE20150643A1 (en) | 2012-06-22 | 2015-05-29 | Cytomx Therapeutics Inc | ANTI-JAGGED 1 / JAGGED 2 CROSS-REACTIVE ANTIBODIES ACTIVABLE ANTI-JAGGED ANTIBODIES AND METHODS OF USE OF THEM |
| WO2014004549A2 (en) | 2012-06-27 | 2014-01-03 | Amgen Inc. | Anti-mesothelin binding proteins |
| US9682143B2 (en) | 2012-08-14 | 2017-06-20 | Ibc Pharmaceuticals, Inc. | Combination therapy for inducing immune response to disease |
| CA2908988A1 (en) | 2013-04-19 | 2014-10-23 | Covagen Ag | Novel bispecific binding molecules with antitumoral activity |
| RU2015156888A (en) | 2013-06-04 | 2017-07-14 | Сайтомкс Терапьютикс, Инк. | COMPOSITIONS AND METHODS OF CONJUGATION OF ACTIVATED ANTIBODIES |
| JP6546160B2 (en) | 2013-06-10 | 2019-07-17 | デイナ ファーバー キャンサー インスティチュート，インコーポレイテッド | Methods and compositions for reducing immunosuppression by tumor cells |
| US11161906B2 (en) | 2013-07-25 | 2021-11-02 | Cytomx Therapeutics, Inc. | Multispecific antibodies, multispecific activatable antibodies and methods of using the same |
| US9822186B2 (en) | 2014-03-28 | 2017-11-21 | Xencor, Inc. | Bispecific antibodies that bind to CD38 and CD3 |
| CA2955947A1 (en) | 2014-07-25 | 2016-01-28 | Cytomx Therapeutics, Inc. | Anti-cd3 antibodies, activatable anti-cd3 antibodies, multispecific anti-cd3 antibodies, multispecific activatable anti-cd3 antibodies, and methods of using the same |
| EP3174901B1 (en) | 2014-07-31 | 2019-06-26 | Amgen Research (Munich) GmbH | Optimized cross-species specific bispecific single chain antibody constructs |
| EP3186277B1 (en) | 2014-08-27 | 2020-10-07 | Memorial Sloan Kettering Cancer Center | Antibodies, compositions, and uses |
| DK3186284T3 (en) | 2014-08-28 | 2022-05-09 | Bioatla Inc | CONDITIONALLY ACTIVE CHIMERIC ANTIGEN RECEPTORS FOR MODIFIED T-CELLS |
| WO2016179003A1 (en) | 2015-05-01 | 2016-11-10 | Genentech, Inc. | Masked anti-cd3 antibodies and methods of use |
| PT3292150T (en) * | 2015-05-04 | 2020-06-01 | Cytomx Therapeutics Inc | Anti-cd166 antibodies, activatable anti-cd166 antibodies, and methods of use thereof |
| SG10202002131PA (en) | 2015-05-21 | 2020-05-28 | Harpoon Therapeutics Inc | Trispecific binding proteins and methods of use |
| EP3337507A4 (en) * | 2015-08-17 | 2019-04-24 | MacroGenics, Inc. | Bispecific monovalent diabodies that are capable of binding b7-h3 and cd3, and uses thereof |
| EP3377301B1 (en) | 2015-11-16 | 2020-09-30 | Fuji Seal International, Inc. | Method and system for forming sleeved containers |
| CN112807429A (en) | 2015-11-19 | 2021-05-18 | 雷维托普有限公司 | Functional antibody fragment complementation of a two-component system for redirected killing of unwanted cells |
| SG11201807548SA (en) | 2016-03-08 | 2018-09-27 | Maverick Therapeutics Inc | Inducible binding proteins and methods of use |
| IL263102B2 (en) | 2016-05-20 | 2023-11-01 | Harpoon Therapeutics Inc | Single domain serum albumin binding protein |
| SG11201810331YA (en) | 2016-05-20 | 2018-12-28 | Harpoon Therapeutics Inc | Single chain variable fragment cd3 binding proteins |
| CN106632681B (en) | 2016-10-11 | 2017-11-14 | 北京东方百泰生物科技有限公司 | Anti- EGFR and AntiCD3 McAb bispecific antibody and its application |
| WO2018071777A1 (en) | 2016-10-14 | 2018-04-19 | Harpoon Therapeutics, Inc. | Innate immune cell trispecific binding proteins and methods of use |
| WO2018160671A1 (en) | 2017-02-28 | 2018-09-07 | Harpoon Therapeutics, Inc. | Targeted checkpoint inhibitors and methods of use |
| US11535668B2 (en) | 2017-02-28 | 2022-12-27 | Harpoon Therapeutics, Inc. | Inducible monovalent antigen binding protein |
| CA3058477A1 (en) | 2017-04-11 | 2018-10-18 | Inhibrx, Inc. | Multispecific polypeptide constructs having constrained cd3 binding and methods of using the same |
| CN111315773A (en) | 2017-09-08 | 2020-06-19 | 马弗里克治疗公司 | Conditionally active binding moieties comprising an Fc region |
| JP7268005B2 (en) | 2017-09-08 | 2023-05-02 | 武田薬品工業株式会社 | Constrained, Conditionally Activated Binding Proteins |
| WO2019222282A1 (en) | 2018-05-14 | 2019-11-21 | Harpoon Therapeutics, Inc. | Conditionally activated binding protein comprising a sterically occluded target binding domain |
| CA3100327A1 (en) | 2018-05-14 | 2019-11-21 | Harpoon Therapeutics, Inc. | Binding moiety for conditional activation of immunoglobulin molecules |
| US20210284728A1 (en) | 2018-05-14 | 2021-09-16 | Harpoon Therapeutics, Inc. | Dual binding moiety |
| WO2020028444A1 (en) | 2018-07-30 | 2020-02-06 | University Of Southern California | Improving the efficacy and safety of adoptive cellular therapies |
| EP3833444A1 (en) | 2018-08-09 | 2021-06-16 | Maverick Therapeutics, Inc. | Coexpression and purification method of conditionally activated binding proteins |
| US20220017626A1 (en) | 2018-09-21 | 2022-01-20 | Harpoon Therapeutics, Inc. | Egfr binding proteins and methods of use |
| AU2019402097A1 (en) | 2018-12-17 | 2021-06-10 | Revitope Limited | Twin immune cell engager |
| CN113603783B (en) | 2018-12-21 | 2022-07-12 | 浙江时迈药业有限公司 | Protease cleavable bispecific antibodies and uses thereof |
| JP2022524338A (en) | 2019-03-05 | 2022-05-02 | 武田薬品工業株式会社 | Restrained and conditionally activated binding protein |
| WO2020181145A1 (en) | 2019-03-05 | 2020-09-10 | Maverick Therapeutics, Inc. | Conditionally activated binding proteins containing fc regions and moieties targeting tumor antigens |
| WO2020223108A1 (en) | 2019-05-02 | 2020-11-05 | The General Hospital Corporation | Teac and attac immunooncology compositions and methods |
-
2018
- 2018-09-06 JP JP2020513686A patent/JP7268005B2/en active Active
- 2018-09-06 IL IL302613A patent/IL302613A/en unknown
- 2018-09-06 IL IL273119A patent/IL273119B2/en unknown
- 2018-09-06 CN CN201880072182.XA patent/CN111356700A/en active Pending
- 2018-09-06 KR KR1020207010104A patent/KR20200047687A/en active IP Right Grant
- 2018-09-06 MY MYPI2020001225A patent/MY201723A/en unknown
- 2018-09-06 PE PE2020000334A patent/PE20212205A1/en unknown
- 2018-09-06 CA CA3075034A patent/CA3075034A1/en active Pending
- 2018-09-06 SG SG11202002089RA patent/SG11202002089RA/en unknown
- 2018-09-06 AU AU2018328291A patent/AU2018328291B2/en active Active
- 2018-09-06 BR BR112020004543-8A patent/BR112020004543A2/en unknown
- 2018-09-06 IL IL302614A patent/IL302614A/en unknown
- 2018-09-06 EP EP18782832.2A patent/EP3679067A2/en active Pending
- 2018-09-06 WO PCT/US2018/049774 patent/WO2019051102A2/en unknown
- 2018-09-06 MX MX2020002667A patent/MX2020002667A/en unknown
- 2018-09-07 TW TW112143878A patent/TW202426489A/en unknown
- 2018-09-07 TW TW112115037A patent/TWI823811B/en active
- 2018-09-07 TW TW107131542A patent/TWI812637B/en active
- 2018-09-07 US US16/124,556 patent/US11406710B2/en active Active
-
2020
- 2020-03-06 CL CL2020000570A patent/CL2020000570A1/en unknown
- 2020-03-09 PH PH12020500474A patent/PH12020500474A1/en unknown
- 2020-04-07 EC ECSENADI202021323A patent/ECSP20021323A/en unknown
- 2020-05-04 ZA ZA2020/02325A patent/ZA202002325B/en unknown
-
2021
- 2021-08-25 CL CL2021002241A patent/CL2021002241A1/en unknown
- 2021-08-25 CL CL2021002242A patent/CL2021002242A1/en unknown
-
2022
- 2022-05-13 US US17/743,995 patent/US11744893B2/en active Active
- 2022-05-13 US US17/743,961 patent/US11744892B2/en active Active
-
2023
- 2023-01-27 AU AU2023200434A patent/AU2023200434A1/en active Pending
- 2023-01-27 AU AU2023200437A patent/AU2023200437A1/en active Pending
- 2023-04-20 JP JP2023069230A patent/JP7490853B2/en active Active
- 2023-07-21 US US18/356,985 patent/US20240139317A1/en active Pending
-
2024
- 2024-05-15 JP JP2024079513A patent/JP2024105561A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007042289A2 (en) * | 2005-10-11 | 2007-04-19 | Ablynx N.V. | Nanobodies™ and polypeptides against egfr and igf-ir |
| WO2011033105A1 (en) * | 2009-09-18 | 2011-03-24 | Micromet Ag | Dosage regimen for administering an epcamxcd3 bispecific antibody |
| WO2013083809A1 (en) * | 2011-12-09 | 2013-06-13 | Amgen Research (Munich) Gmbh | Prevention of adverse effects caused by epcamxcd3 bispecific antibodies |
| WO2016046778A2 (en) * | 2014-09-25 | 2016-03-31 | Amgen Inc | Protease-activatable bispecific proteins |
| WO2016156570A1 (en) * | 2015-04-02 | 2016-10-06 | Ablynx N.V. | Bispecific cxcr4-cd-4 polypeptides with potent anti-hiv activity |
| WO2016180969A1 (en) * | 2015-05-13 | 2016-11-17 | Ablynx N.V. | T cell recruiting polypeptides based on tcr alpha/beta reactivity |
Also Published As
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| IL273119B1 (en) | Constrained conditionally activated binding proteins | |
| AU2020203910B2 (en) | Matrix Metalloproteinase Substrates And Other Cleavable Moieties And Methods Of Use Thereof | |
| IL261432B2 (en) | Inducible binding proteins and methods of use | |
| US20200347132A1 (en) | Constrained conditionally activated binding proteins | |
| IL274892A (en) | Bispecific cd33 and cd3 binding proteins | |
| IL281970A (en) | Methods of using bispecific cd33 and cd3 binding proteins | |
| IL300598A (en) | Constrained conditionally activated binding proteins | |
| US12128102B2 (en) | Constrained conditionally activated binding proteins | |
| EA045012B1 (en) | LIMITED CONDITIONALLY ACTIVATE BINDING PROTEINS | |
| KR20230165798A (en) | Treatment Methods Using Constrained Conditional Activation Binding Proteins |